Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:4.6.1.2 (guanylate cyclase)
8,497 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Soluble guanylyl cyclase (sGC) is the major physiological target of sydnonimine-based vasodilators such as molsidomine. Decomposition of sydnonimines results in the stoichiometric formation of nitric oxide (NO) and superoxide (O2-), which rapidly react to form peroxynitrite. Inasmuch as sGC is activated by NO but not by peroxynitrite, we investigated the mechanisms underlying sGC activation by 3-morpholinosydnonimine (SIN-1). Stimulation of purified bovine lung sGC by SIN-1 was found to be strongly dependent on glutathione (GSH). By contrast, GSH did not affect sGC activation by NO released from 2,2-diethyl-1-nitroso-oxyhydrazine, indicating that NO/O2- released from SIN-1 converted GSH to an activator of sGC. High performance liquid chromatography identified this product as the thionitrite S-nitrosoglutathione. Further, the reaction product decomposed to release NO upon addition of Cu(NO3)2 in the presence of GSH. Activation of sGC was antagonized by the Cu(I)-specific chelator neocuproine, whereas the Cu(II)-selective drug cuprizone was less potent. Carbon dioxide (delivered as NaHCO3) antagonized S-nitrosation by peroxynitrite but not by SIN-1. Thus, NO/O2- released from SIN-1 mediates a CO2-insensitive conversion of GSH to S-nitrosoglutathione, a thionitrite that activates sGC via trace metal-catalyzed release of NO. These results may provide novel insights into the molecular mechanism underlying the nitrovasodilator action of SIN-1.
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PMID:Activation of soluble guanylyl cyclase by the nitrovasodilator 3-morpholinosydnonimine involves formation of S-nitrosoglutathione. 965 7

Peroxynitrite (ONOO-), a potent oxidant formed by reaction of nitric oxide (NO.) with superoxide anion, can activate guanylyl cyclase and is able to induce vasodilation or inhibit platelet aggregation and leukocyte adhesion, via thiol-dependent formation of NO. Reaction of ONOO- with thiols is thought to proceed through formation of a S-nitrothiol (thionitrate; RSNO2) intermediate and yields low levels of S-nitrosothiols (thionitrites; RSNO), both of which are theoretical sources of NO. Kinetic analysis of NO. production after reaction of ONOO- with GSH established that NO. originates exclusively from the thionitrite GSNO. Further mechanistic investigations indicated that GSNO formation by ONOO- does not occur via one-electron oxidation mechanisms. Nitrosation of GSH could theoretically proceed via intermediate formation of the thionitrate GSNO2, which, after rearrangement to the corresponding sulfenyl nitrite (GSONO), can react with GSH to form GSNO and GSOH. However, no evidence for such a mechanism was found in experiments with NO2. or with the stable nitrothiol tert-butylthionitrate. Using high performance liquid chromatography with chemiluminescence detection, formation of H2O2 was observed after reaction of ONOO- with GSH under both aerobic and anaerobic conditions, at levels similar to the yield of GSNO, indicative of a direct nucleophilic nitrosation mechanism with elimination of HOO-. Our results indicate that ONOO- may contribute to S-nitrosation in vivo and that direct nitrosation of thiols or other nucleophilic substrates by ONOO- may represent an important and often overlooked component of NO. biochemistry.
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PMID:Formation of S-nitrosothiols via direct nucleophilic nitrosation of thiols by peroxynitrite with elimination of hydrogen peroxide. 980 85

Soluble guanylate cyclase (sGC) catalyzes the conversion of GTP to cGMP and is activated several hundred-fold by binding of nitric oxide (*NO) to the heme prosthetic group. We have examined the stability of the nitrosyl-heme complex of sGC (*NO-sGC) at 37 degreesC in order to determine whether simple dissociation of *NO from sGC could account for the observed in vivo deactivation time. Recombinant sGC was purified from Sf9 cells coinfected with baculoviruses containing the cDNAs for the alpha1 and beta1 subunits of rat lung sGC. The purified protein contained a stoichiometric equivalent of ferrous high-spin heme. Characterization of the purified protein found it to be essentially identical to that purified from bovine lung. Ferrous-nitrosyl sGC prepared anaerobically and exchanged into aerobic buffer containing no reducing agents was essentially stable on ice and had a half-life of approximately 90 min at 37 degreesC. In the presence of thiols [DTT, glutathione (GSH), or L-cysteine], *NO was rapidly lost from sGC regenerating the ferrous high-spin form of the heme. The half-life of *NO-sGC in the presence of 1 mM GSH at 37 degreesC was 6.3 min. In the presence of oxyhemoglobin, the half-life was further reduced to 2.9 min. Although these rates are not fast enough to account for that observed in vivo, and thus probably involve additional agent(s), these data do imply a role for low molecular weight thiols, such as GSH, and oxyferrohemoproteins, such as oxymyoglobin, in the deactivation of sGC.
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PMID:Regeneration of the ferrous heme of soluble guanylate cyclase from the nitric oxide complex: acceleration by thiols and oxyhemoglobin. 983 82

Our previous work suggests that relaxation of endothelium-removed bovine coronary arteries (BCA) to posthypoxic reoxygenation is mediated by NADH oxidase-dependent superoxide anion-derived H2O2 and cGMP. The purpose of this study was to investigate if altering BCA GSH peroxidase activity by enhancing its activity with a GSH peroxidase-mimetic (0.1 mM Ebselen) or by inhibiting its activity with an inhibitor of GSH peroxidase [10 mM mercaptosuccinic acid (MS)] causes a selective modulation of responses to exogenously (1 microM-1 mM H2O2) and endogenously generated (reoxygenation and 1-10 mM lactate) H2O2. Ebselen inhibited and MS enhanced all of the responses that are thought to be mediated by H2O2, without having significant effects on relaxation to hypoxia or a nitric oxide donor [1 nM-10 microM S-nitroso-N-acetylpenicillamine (SNAP)]. Thus enhancement of BCA GSH peroxidase activity with Ebselen inhibits relaxation to reoxygenation, lactate, and H2O2, whereas inhibition of GSH peroxidase with MS causes potentiation of responses thought to be mediated by H2O2 in BCA. Inactivation of catalase by pretreatment of BCA with 3-amino-1,2,4-triazole (50 mM, 30 min) inhibited relaxation to H2O2 and the potentiation by MS. Whereas the actions of these probes are not consistent with a role for oxidation of GSH in the relaxation to H2O2, their effects are potentially a result of modulating the metabolism of H2O2 by endogenous catalase, which is thought to mediate the stimulation of the cytosolic or soluble form of guanylate cyclase.
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PMID:Influence of glutathione peroxidase on coronary artery responses to alterations in PO2 and H2O2. 988 37

Nitric oxide-selective sensors have been prepared with the heme domain of soluble guanylate cyclase (sGC), the only known receptor for signal transduction involving nitric oxide. Expressed in and purified from E. coli, the heme domain contains a stoichiometric amount of heme that has electronic and resonance Raman spectra almost identical to those of heterodimeric (native) sGC purified from bovine lung. The small size of the heme domain, its inability to bind oxygen, and its high affinity for nitric oxide make it well-suited for sensor applications. The heme domain has been labeled with a fluorescent reporter dye and changes in this dye's intensity are observed based on the sGC heme domain's characteristic binding of nitric oxide. The current sensors are prepared with 100-microns optical fiber but could also be prepared using submicrometer fiber tips. These sensors have fast, linear, and reversible responses to nitric oxide and are unaffected by numerous common interferents, such as oxygen, nitrite and nitrate. The sensor limit of detection is 1 microM nitric oxide. Glutathione has been shown to decrease the sensitivity of the sensor; however, the sensor response remains linear and can be calibrated on the basis of the glutathione concentration present in the biological environment of interest. The sensors have been used to measure extracellular nitric oxide production by BALB/c mouse macrophages. Minimal nitric oxide was produced by untreated cells, while high levels of nitric oxide were released from activated cells, e.g., 111 +/- 2 microM in a given cell culture.
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PMID:Cellular applications of a sensitive and selective fiber-optic nitric oxide biosensor based on a dye-labeled heme domain of soluble guanylate cyclase. 1036 89

Soluble guanylyl cyclase (sGC) is an alpha/beta-heterodimeric hemoprotein that, upon interaction with the intercellular messenger molecule NO, generates cGMP. Although the related family of particulate guanylyl cyclases (pGCs) forms active homodimeric complexes, it is not known whether homodimerization of sGC subunits occurs. We report here the expression in Sf9 cells of glutathione S-transferase-tagged recombinant human sGCalpha1 and beta1 subunits, applying a novel and rapid purification method based on GSH-Sepharose affinity chromatography. Surprisingly, in intact Sf9 cells, both homodimeric GSTalpha/alpha and GSTbeta/beta complexes were formed that were catalytically inactive. Upon coexpression of the respective complementary subunits, GSTalpha/beta or GSTbeta/alpha heterodimers were preferentially formed, whereas homodimers were still detectable. When subunits were mixed after expression, e.g. GSTbeta and beta or GSTalpha and beta, no dimerization was observed. In conclusion, our data suggest the previously unrecognized possibility of a physiological equilibrium between homo- and heterodimeric sGC complexes.
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PMID:Homodimerization of soluble guanylyl cyclase subunits. Dimerization analysis using a glutathione s-transferase affinity tag. 1037 11

Recent results demonstrated that S-nitrosoglutathione (GSNO) and nitric oxide (*NO) protect brain dopamine neurons from hydroxyl radical (*OH)-induced oxidative stress in vivo because they are potent antioxidants. GSNO and *NO terminate oxidant stress in the brain by (i) inhibiting iron-stimulated hydroxyl radicals formation or the Fenton reaction, (ii) terminating lipid peroxidation, (iii) augmenting the antioxidative potency of glutathione (GSH), (iv) mediating neuroprotective action of brain-derived neurotrophin (BDNF), and (v) inhibiting cysteinyl proteases. In fact, GSNO--S-nitrosylated GSH--is approximately 100 times more potent than the classical antioxidant GSH. In addition, S-nitrosylation of cysteine residues by GSNO inactivates caspase-3 and HIV-1 protease, and prevents apoptosis and neurotoxicity. GSNO-induced antiplatelet aggregation is also mediated by S-nitrosylation of clotting factor XIII. Thus the elucidation of chemical reactions involved in this GSNO pathway (GSH GS* + *NO-->[GSNO]-->GSSG + *NO-->GSH) is necessary for understanding the biology of *NO, especially its beneficial antioxidative and neuroprotective effects in the CNS. GSNO is most likely generated in the endothelial and astroglial cells during oxidative stress because these cells contain mM GSH and nitric oxide synthase. Furthermore, the transfer of GSH and *NO to neurons via this GSNO pathway may facilitate cell to neuron communications, including not only the activation of guanylyl cyclase, but also the nitrosylation of iron complexes, iron containing enzymes, and cysteinyl proteases. GSNO annihilates free radicals and promotes neuroprotection via its c-GMP-independent nitrosylation actions. This putative pathway of GSNO/GSH/*NO may provide new molecular insights for the redox cycling of GSH and GSSG in the CNS.
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PMID:The redox pathway of S-nitrosoglutathione, glutathione and nitric oxide in cell to neuron communications. 1063 Jun 87

The discoveries of physiological roles of nitric oxide (.NO) as the mediator of endothelium-derived relaxing factor (EDRF) action and the activator of guanylyl cyclase to increase cyclic guanosine monophosphate (cGMP), which lead to vasorelaxation in the cardiovascular system, have been awarded with the 1998 Nobel Prize of Medicine. The present review discusses putative beneficial effects of .NO in the central nervous system (CNS). In addition to its prominent roles of the regulation of cerebral blood flow and the modulation of cell to cell communication in the brain, recent in vitro and in vivo results indicated that .NO is a potent antioxidative agent. .NO terminates oxidant stress in the brain by (i) suppressing iron-induced generation of hydroxyl radicals (.OH) via the Fenton reaction, (ii) interrupting the chain reaction of lipid peroxidation, (iii) augmenting the antioxidative potency of reduced glutathione (GSH) and (iv) inhibiting cysteine proteases. It is apparent that .NO--a relative long half-life nitrogen-centered weak radical--scavenges those short-lived, highly reactive free radicals such as superoxide anion (O2.-), .OH, peroxyl lipid radicals (LOO.) and thiyl radicals (i.e., GS.), yielding reactive nitrogen species including nitrites, nitrates, S-nitrosoglutathione (GSNO) and peroxynitrite (ONOO-). GSNO is 100-fold more potent than GSH; it completely inhibits the weak peroxidative effect of ONOO-. Moreover, CO2 and .NO neutralize prooxidative effects of ONOO-. CO2 prevents protein oxidation but not 3-nitrotyrosine formation caused by ONOO-. Finally, neuroprotective effects of GSNO and .NO have been demonstrated in brain preparations in vivo. These novel neuroprotective properties of .NO and GSNO may have their physiological significance, since oxidative stress depletes GSH while increasing GS. and .NO formation in astroglial and endothelial cells, resulting in the generation of a more potent antioxidant GSNO and providing additional neuro-protection at microM concentrations. This putative GSNO pathway (GSH-->GS.-->GSNO-->.NO + GSSG-->GSH) may be an important part of endogenous antioxidative defense system, which could protect neurons and other brain cells against oxidative stress caused by oxidants, iron complexes, proteases and cytokines. In conclusion, .NO is a potent antioxidant against oxidative damage caused by reactive oxygen species, which are generated by Fenton reaction or other mechanisms in the brain via redox cycling of iron complexes.
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PMID:Neuroprotective properties of nitric oxide. 1066 35

Dopamine-beta-hydroxylase (DbetaH) is a copper-containing enzyme that uses molecular oxygen and ascorbate to catalyze the addition of a hydroxyl group on the beta-carbon of dopamine to form norepinephrine. While norepinephrine causes vasoconstriction following reflex sympathetic stimulation, nitric oxide (NO) formation results in vasodilatation via a guanylyl cyclase-dependent mechanism. In this report, we investigated the relationship between NO and DbetaH enzymatic activity. In the initial in vitro experiments, the activity of purified DbetaH was inhibited by the NO donor, diethylamine/NO (DEA/NO), with an IC(50) of 1 mm. The inclusion of either azide or GSH partially restored DbetaH activity, suggesting the involvement of the reactive nitrogen oxide species, N(2)O(3). Treatment of human neuroblastoma cells (SK-N-MC) with diethylamine/NO decreased cellular DbetaH activity without affecting their growth rate and was augmented by the depletion of intracellular GSH. Co-culture of the SK-N-MC cells with interferon-gamma and lipopolysaccharide-activated macrophages, which release NO, also reduced the DbetaH activity in the neuroblastoma cells. Our results are consistent with the hypothesis that nitrosative stress, mediated by N(2)O(3), can result in the inhibition of norepinephrine biosynthesis and may contribute to the regulation of neurotransmission and vasodilatation.
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PMID:Inhibitory effects of nitric oxide and nitrosative stress on dopamine-beta-hydroxylase. 1088 4

The nitric oxide (NO) donor, S-nitroso-N-acetyl-D,L-penicillamine (SNAP), induced differentiation of human neuroblastoma NB69 cells to a dopamine phenotype, as shown by phase-contrast microscopy and tyrosine hydroxylase (TH) immunocytochemistry. NB69 cells were treated with 50 to 750 microM SNAP in serum-free-defined medium for 24 h. SNAP treatment did not increase the number of necrotic or apoptotic cells. However, a decrease in the number of viable cells was observed at 750 microM SNAP. In addition, a decrease in (3)H-thymidine uptake was detected at the highest dose of SNAP. An increase in the antiapoptotic Bcl-2 and Bcl-xL protein levels and a decrease in the proapoptotic Bax and Bcl-xS protein levels were also detected by Western blot analysis after SNAP treatment. At low doses (50-125 microM), SNAP induced an increase in catecholamine levels, (3)H-dopamine uptake, TH activity and monoamine metabolism, while a decrease in all these parameters was observed at high doses (250-750 microM). The TH protein content, analyzed by Western blot, remained unchanged in SNAP-treated cells throughout the range of doses studied, when compared with the control group. SNAP produced a dose-dependent decrease in the glutathione (GSH) content of the culture medium, without altering intracellular GSH. In addition, cGMP levels and nitrite concentration, measured in the supernatant of SNAP-treated cells, increased in a dose-dependent manner, as compared to control levels. The guanylate cyclase inhibitor lH-[1,2, 4]oxadiazolo[4,3a]quinoxaline-l-one (ODQ) did not revert the SNAP-induced effect on (3)H-dopamine uptake to control values. These results suggest that NO, released from SNAP, induces differentiation of NB69 cells and regulates TH protein at the post-transcriptional level through a cGMP-independent mechanism.
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PMID:Nitric oxide induces differentiation in the NB69 human catecholamine-rich cell line. 1096 52


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